Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre- mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events - which are not understood mechanistically - will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which altered spliceosomal dynamics impact splicing fidelity. Experiments will investigate contributions of the U1-U2 protein-interaction network and the ATPase Prp5 to intron definition and to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a unique commitment to spliceosome assembly and a simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the binding site of the 3' splice site substrate for the second step of splicing, which will use an 'orthogonal spliceosome' system in yeast, and on the mechanism by which the second-step substrate replaces first-step product in the catalytic core. Finally, we are investigating the role of novel RNA-RNA interactions in the transition from the first-to-second step of splicing. Together, the models proposed in these aims will greatly improve our understanding of the dynamic range of RNA-RNA interactions at consecutive stages of splicing.